Translation of abstract (English)

Melanoma cells that primarily arise from a malignant transformation of pigment-producing cells of the skin, can be selectively destroyed by autologous CD8+ T lymphocytes. The selectivity is provided by the interaction between the T cell receptor (TCR) and specific tumor cell surface complexes, consisting of antigenic peptides bound to HLA class I molecules. The antigenic peptides originate from the degradation of tumor proteins. In general, the multi-catalytic proteasome complex initiates the cleavage of tumor antigens into peptide fragments of different length, thereby defining the C-terminus for the majority of peptides presented by HLA class I molecules. While some of the proteasome products are of the correct size for direct HLA class I binding others are N-terminally elongated peptide precursors that require further trimming by aminopeptidases. Currently two human ER-localized aminopeptidases are known, ERAP1 and ERAP2. While ERAP1 is known to be of specific relevance for the efficient generation of viral and parasitic antigenic peptides, the importance of the peptidase in processing tumor-associated peptide epitopes has not been determined. Thus, the aim of this thesis was to determine the role of ERAP1 in antigen presentation by melanoma cells. Furthermore stimuli and signaling pathways that regulate ERAP1 and ERAP2 expression should be defined. To analyze the impact of ERAP1 on antigen presentation of melanoma cells, the autologous tumor-CD8+ T cell system of melanoma patient Ma-Mel-86 was selected. In the cell line Ma-Mel-86a and Ma-Mel-86c, established from different metastasis of this patient, ERAP1 expression was stably downregulated by shRNA. Importantly, ERAP1 knockdown did not influence the overall of HLA class I surface expression level. To determine the effect of reduced ERAP1 expression in tumor cells on antigen presentation, autologous CD8+ T cells derived from peripheral blood were initially stimulated for several times with Ma-Mel-86a cells. Subsequently, the bulk CD8+ T cells were analyzed for their capability to recognize Ma-Mel-86a in comparison to ERAP1 shRNA transfected Ma-Mel-86a melanoma cells. Interestingly ERAP1 downregulation impaired the recognition of Ma-Mel-86a melanoma cells by autologous bulk CD8+ T lymphocytes, suggesting that reduced ERAP1 activity led to an alteration of the antigenic epitope repertoire presented by HLA class I molecules. Diminished ERAP1 expression also decreased the recognition of the tumor cells by an autologous CD8+ T cell clone recognizing a mutated target antigen. Comparable observations were made for Ma-Mel-86c ERAP1 shRNA transfectants, which exhibited reduced stimulatory capacity towards two of three autologous CD8+ T cell clones of different specificity. Due to the important role of ERAP1 in epitope presentation by melanoma cells the expression profile of ERAP1 and ERAP2 as well as their regulation in several cell lines was analyzed. Firstly, strong distinctions in ERAP1 and ERAP2 expression could be detected between cell lines established from different metastasis of melanoma patients. Since chemotherapeutic drugs, like Doxorubicin, are currently under intense investigation for their immune modulating properties its impact on ERAP1 and ERAP2 expression was determined. Interestingly, the treatment of several cell lines with Doxorubicin led to an increase in promoter activity as well as mRNA expression of both aminopeptidases, with ERAP1 expression being dependent of the p53 status of the cells. However the elevated expression was only partially detectable on the protein level, pointing towards additional post-transcriptional regulation of ERAP1 and ERAP2 expression. The results of this thesis demonstrate that ERAP1 plays an important role in the generation of tumor-associated CD8+ T cell epitopes in melanoma cells. Thus a heterogeneous ERAP expression as it can be detected in metastasis of tumor patients might be associated with quantitative and qualitative alterations in the antigenic HLA class I peptide repertoire. This effect could influence the efficiency of epitop-specific tumor therapies such as the adoptive transfer of TCR-transgenic CD8+ T cells and should be taken into consideration for therapy design.